6.2.1e-i Biotechnology Flashcards
Define biotechnology
-the industrial use of living organisms, or parts of living organisms to produce food, drugs, or other products
( e.g. yogurt, bread, beer) for human benefit
Define microorganism
a microscopic organism - e.g. bacteria, fungi, yeast
What are the reasons why microorganisms are useful for biotechnological processes? (6)
1- no welfare issues
2- easily genetically engineered to carry out specific reactions
3- have rapid growth rate in favourable conditions
4- grow well at low temperatures (compared to non biological processes) - making it cheap
5- all conditions for optimum growth can be met in a fermenter
6- can feed on waste useless/toxic waste products from other reactions
What are the 2 categories of food production using microorganisms?
Direct and indirect
What are the 4 indirect processes that use microorganisms?
- Cheese making
- Baking
- Brewing
- Yogurt making
What is the difference between direct and indirect food production?
indirect: A —-> B using microorganism (M)
e.g. flour to bread using yeast
direct: M —–> C microorganism to product
e.g. - fungus to quorn
Describe the indirect process of brewing
1 - yeast respires anaerobically using glucose from grains
2- produces CO2 and ethanol
Describe the indirect process of baking
1- yeast respires aerobically and feeds on nutrients
2- yeast produces CO2 which causes bread to rise
Describe the indirect process of cheese making
1- chymosin enzyme gained from rennet or GM yeast and lactic acid bacteria
2- chymosin clots milk
3- bacteria convert lactose into lactic acid - sour + solidifying
Describe the indirect process of yoghurt making
1- lactobacillus bacteria converts lactose in milk to latic acid
2- lactic acid clots milks and thickens it and lowers pH - sour
What is one example of a microorganism being used to directly to make food for human consumption?
Single cell protein (SCP)
- best known SCP is QUORN (made from Fungus fusarium)
What are the advantages of using microorganisms to make human food?
1- microorganisms reproduce fast
2- microorganisms produce protein faster than animals and plants
3- microorganisms have high protein content with little fat
4- microorganisms can use wide variety of waste materials incl human and animal waste - reduce costs
5- microorganisms can be genetically modified to produce protein required
6- microorganism growth not dependant on seasons - takes place throughout year to meet demand
7- no welfare issues
8- can be made to taste like anything
What are the disadvantages of using microorganisms to make human food?
1- some microorganisms can produce toxins if optimum conditions not maintained
2- microorganisms have to be seperated from culture medium and processed to make the food
3- need carefully controlled sterile conditions - add to costs
4- some people object to use of genetically modified (GM) organisms in food
5- protein has to be purified to ensure no toxins/contaminants
6- many people dislike thought of eating microorganisms grown on waste
7- has little natural flavour - additives needed
Describe how penicillin is produced commercially?
1- fungi from Penicillium genus is grown under stress in an industrial fermenter (nutrients added throughout)
2- under stress fungi from Penicillin genus produces antibiotic penicillin which stops bacteria from growing and competing for resources
What is the name of the microorganism used to produce penicillin?
fungi from Penicillium genus
e.g. Penicillium chrysogenum
Describe how human insulin can be produced on a large scale using biotechnology
1- insulin is made from GM (genetically modified) bacteria
2- gene for human insulin is inserted into their DNA
3- this bacteria is grown in a large scale industrial fermenter
4- insulin produced is purified
Define bioremediation
the use of microorganisms to break down pollutants/contaminants in soil or water
What are the 2 different approaches to bioremediation?
- using natural organisms
supporting them with extra nutrients and enhanced growing conditions to encourage the process - using GM organisms
genetically modified bacteria able to break down toxins they would not normally encounter
What are the 2 ways of culturing microorganisms in a lab?
describe them
to culture microorganisms you need a nutrient medium (food)
1- broth (liquid medium)
microorganisms grow throughout the volume
- so much higher populations
- can stir to redistribute nutrients
- easy transfer by pouring
2- agar (solid medium)
-good for counting and isolating colonies so used for experiments and studying
Define contaminant
any unwanted microorganism
Define aseptic technique
the measures taken to minimise the risk of contaminants entering cultures of microorganisms
Define asepsis
the absence of unwanted microorganisms
What are 3 aspectic techniques used at laboratory and starter culture level?
describe them
1- loop in bunsen flame before inoculation
2- bunsen flame to draw air up away from culture
3- minimise opening time and only partially open the lid
What are 3 aspects techniques used at large-scale culture level?
describe them
1- steam - sterilises fermenter between batches
2- nutrient medium sterilised to prevent addition of contaminants
3- stainless steel
4- filters
5- minimise additions to culture
What are 5 reasons why contaminants are a problem in industrial fermentation?
1- they may compete with culture microorganisms for nutrients and space
2- they may reduce the yield of useful products from the culture
3- they may spoil the product
4- they may produce toxic chemicals
5- may destroy the culture microorganisms and their products
Define fermentation
the culturing of microorganisms both aerobically and anaerobically in fermentation tanks to produce any useful substance
Define culture
a growth of microorganisms
Define pure culture
a growth of microorganisms consisting of a single species
Define mixed culture
a growth of microorganisms consisting of several species
Define closed culture
the growth of microorganisms in an environment where all conditions are fixed and contained
- no new materials are added and no waste products of organisms are removed
Draw, label and annotate a grpah of the standard growth curve for microorganisms in a closed culture
diagram on paper flashcard
on paper flashcard
1- lag phase - pop increase slow, reproduction rate low
population increasing slowly as microorganisms are adapting to environment and synthesising enzymes
2- exponential/log phase - pop increase quick/at maximum
culture conditions at optimum for reproduction (lots food/ low competition + enough space)
3- stationary phase - death rate = reproductive rate
not enough food, waste products build up
4- decline phase - death rate greater than reproductive rate
not enough food, waste products at toxic levels
How would adding nutrients or removing waste products during different phases affect the shape of the standard growth curve?
to exponential/lag phase:
- adding nutrients/removing waste:
extends the population size so stationary phase starts at a higher population level (no effect on gradient)
to stationary phase:
- adding nutrients/removing waste:
extends the phase if decline is due to nutrients running out/waste build up
to decline phase:
-adding nutrients: slow the decline if due to nutrients running out
- removing waste: slow the decline if there are enough nutrients
What are 5 factors that may prevent exponential growth in a culture of bacteria? explain for each why they become limiting
1) nutrients available e.g. glucose/respiratory substrate
-nutrient level insufficient to support further growth and reproduction as it is used up by microorganisms
2) build up of toxic waste
-bacteria number rise= toxic material build up inhibit further growth or kill culture
3) temperature
-enzymes denature if too high
-low temperature slows growth + reproduction
4) pH
-effects enzyme activity and inhibits population growth
5) oxygen levels
- as population rises=oxygen demand rise for aerobic respiration
Describe a step by step method to investigate the effect of one factor on the growth of microorganisms.
(nutrients/pH/temperature)
either
a) set up identical colonies in different conditions of temperature
b) set up serial dilutions of nutrients/pH at a set temperature
e.g temperature
1) Using a pipette add a set volume (e.g. 0.1cm^3) of sample of bacteria in broth to an agar plate
2) Spread the broth across the entire surface of the agar using a sterile plastic spreader
3) Put lid on agar plate and lightly tape it shut
4) Repeat steps 1-3 to have 6 plates in total
5) place 3 plates in fridge at 4°C and 3 in incubator at 25 °C
if no incubator leave at room temperature in room with constant temp
6) incubate plates upside down to stop condensation forming on lid and dropping into agar
7) negative controls: place 2 lidded uncultured agar plates in each location
8) leave all plates incubated for same amount of time
9) count number of colonies of bacteria on surface of agar
10) work mean number of colonies at each temperature
What are the 2 main ways of growing microorganisms called (in vessels) ?
Batch Fermentation and Continuous Fermentation
Explain batch fermentation
- microorganisms are grown in individual batches in a fermentation vessel
- when one culture ends its removed and a different batch of microorganisms is grown in the vessel
- no nutrients added
- tank cleaned
What are the large containers in which cultures are grown in called?
Fermentation vessels
Explain continuous fermentation
- microorganisms are continually grown in a fermentation vessel without stopping
- nutrients are replenished at constant rate
- waste products are removed at constant rate
Define metabolite
a chemical produced by a cells metabolism
Define Primary metabolite
a substance produced by an organism as part of its normal cell cycle
Define Secondary metabolite
a substance produced by an organism that is not part of its normal cell cycle.
Secondary metabolites are produced when cells are under stress or beecause of competition in an intense environment
Are primary and secondary metabolites produced by all microorganisms or only some?
Primary metabolites: are produced by all
Secondary metabolities: only produced by some
Compare the advantages and disadvantages of batch and continuous culture?
1) Growth rate:
batch - has lag-log-stationary phase
continuous - has optimum rate maintained
2) Ease of set up and maintenance:
batch - easy to set uo and run
continuous - not easy , requires sophisticated equipment and constant monitoring and adjustment
3) Consequence of contamination:
batch- less chance of contamination, 1 batch lost, sterilise and start again
continuous - more chance of contamination, most cost to restard as whole culture lost
4) Efficiency:
batch - lower , time lost between batches
continuous - higher
5) good for primary or secondary metabolites?
batch can be altered for either by changing factors
continuous good for primary
Draw a graph to show how the production of primary and secondary metabolites changed over the time of the standard growth curve
diagram on paper flashcard
-primary metabolites produced in periods of active growth
-secondary metabolities tend to be produced in stationary phase
Why does the production of primary metabolites in a microorganism match the overall growth of the microorganism population?
- primary metabolities are produced as part of the normal growth of the organism
so all cells produce them - as more cells are produced more metabolites are
Suggest why secondary metabolites such as antibiotics are only produced after the main growth phase of a microorganism
- secondary metabolites tend to be produced under stress so in stationary phase
- in stationary phase resources are limiting the population, there is more competition and stress causing secondary metabolites to be produced
- antibiotics produced to try and reduce competition for resources from bacteria i.e. in stationary phase when the resources are limited
- (in main growth phase there is excess of resources)
Draw and label a diagram of a large scale industrial fermenter
How can an industrial fermenter change or control the growing conditions inside it? explain
1) temperature
-temperature probe and cooling water jacket surrounding entire vessel
- maintain optimum temp so no unwanted toxins produced
- allow enzymes to work efficiently
2) pH
- pH probe and kept at optimum
- allow enzymes to work efficiently
3) Oxygen concentration
- sterile air pumped into vessel when needed
- O2 always available for respiration
4) Nutrients
- paddles circulate nutrient medium constantly around vessel
- avoid it becoming limiting factor
Why does temperature, type and time of addition of nutrient, oxygen concentration, and pH need to be carefully controlled in an industrial fermenter? explain
1) temperaure and pH - affect enzyme reactions and too far from optimum causes toxin production
2) nutrients - need to be added at right time to prevent it becoming limiting factor
and avoid culture going into stationary phase producing unwanted secondary metabolities
3) oxygen concentration - maintain for aerobic respiration
What are the advantages of using isolated enzymes instead of whole organisms?
1) Less wasteful/more efficient
- whole microorganisms use up substrate to grow and reproduce (produce biomass not product)
2) More specific
- no unwanted enzymes present unlike in whole organism
- so no side waste products
3) maximise efficiency
- single enzymes can be given optimum conditions for maximum product formation unlike if whole organism used (conditions must be suitable for growth of whole organism)
4) Less downstream processing
- isolated enzymes produce pure product no seperation needed
- whole organisms produce varierty of products so expensive and difficult to seperate
What are isolated enzymes?
-isolated enzymes become mixed in with the products of a reaction
- products then need to be seperated from the mixture
Define Downstream Processing
The processing required after synthesising a product
(e.g. seperation, purification)
Why do extracellular enzymes tend be used more than intercellular enzymes in biotechnology?
1) extracellular enzymes are secreted - easy to isolate and use
2) Easy to identify and isolate bc only a few extracellular enzymes produced by microorganisms
3) extracellular enzymes more robust (strong)
as conditions outside cell less tightly controlled
they can adapt to cope in variations in pH/temp
Define immobilised enzymes
enzymes which are attatched to an insoluble material preventing them from freely mixing with the solution
What are the advantages of using immobilised enzymes?
1) can be reused - cheaper
2) less down stream processing - cheaper
prevents contamination of product with enzymes - no time spent seperating
3) more stable - cheaper to run
more tolerant of higher temps/pH, less easily denatured
What are the disadvantages of using immobilised enzymes?
1) reduced efficiency/lower rate of reaction
cant freely mix with their substrate
2) higher initial costs of materials
immobilised enzymes more expensive than free enzymes/microorganisms
3) higher initial costs of bioreactor
more expensive fermenter
4) more technical issues
as more complex fermenters used
What are the 4 ways in which enzymes can be immobilised called?
1) adsorption to inorganic carrier
2) entrapment in a matrix
3) covalent/ionic bonds to inorganic carrier
4) encapsulation
Describe the method of immobilsing enzymes: adsorption
Adsorption to inorganic carrier:
- polar molecules bind weakly to a large insoluble matrix (hydrogen bonds)
adv- simple/cheap , used with many different processes, enzymes v.accessible to substrate+activity unchanged
disad- enzymes can be lost from matrix easily
Describe the method of immobilsing enzymes: covalent/ionic bonding
Covalent or ionic bonding to inorganic carrier:
- strong bonds to large insoluble matrix
adv - varied costs , enzymes strongly bound unlikely to be lost, enzymes v.accessible to substrate, pH and substrate conc have little effect on enzyme activity
disad- varied costs, strong bonds may slightly change shape of active site -less effective
Describe the method of immobilsing enzymes: entrapment
Entrapment in matrix:
enzymes trapped in a silica gel matrix, substrates have to diffuse to active site and products from active site
adv- widely applicable to different processes
disad - expensive, can be difficult to entrap , diffusion can be slow,
Describe the method of immobilsing enzymes: encapsulation
Encapsulation:
membrane entrapment in microcapsules e.g. jelly like alginate beads , substrates have to diffuse to active site and products from active site
adv- simple to do, small effect on enzyme activity, applicable to different processes
disad- expensive, diffusion can be slow
which immobilisation methods may affect the shape of an enzymes active site? explain why
Adsorption and Covalent/ionic bonding to inorganic carrier
- bonds can cause shape of the molecule to change
- if bonds formed close to the active site it can alter its shape
Which immobilisation methods may affect the rate at which substrate molecules can gain access to the active sites of enzymes? explain why
Entrapment and Encapsulation
- there is a physical barrier through which the medium much diffuse to reach the enyzmes
- (substrate must diffuse through to active site)
Draw a table to compare the 4 different ways in which enzymes can be immobilised
on paper flashcard
on paper flashcard
What are the 5 examples of the use of immobilised enzymes?
1) immobilised Penicillin Acylase
2) immobilised glucose isomerase
3) immobilised lactase
4) immobilised aminoacylase
5) immobilised glucoamylase
Describe what the immobilised enzyme Penicillin Acylase does.
FORMATION OF:
semi-sythetic penicillins
Describe what the immobilised enzyme Glucose Isomerase does.
glucose isomerase - converts glucose into fructose
(fructose used as sweetener in food)
Describe what the immobilised enzyme lactase does.
lactase - converts lactose into glucose and galactose
(produce lactose free milk)
Describe what the immobilised enzyme aminoacylase does.
aminoacylase - converts a mixture of isomers into pure samples of L-amino acids
(2 amino acids present L and D, body only utilises L amino acid)
Describe what the immobilised enzyme glucoamylase does.
glucoamylase - converts dextrins (carbohydrate products) to glucose
